Scaffold for growing neuronal cells and tissue

ABSTRACT

The present invention provides a composition based on poly-l-lactic acid (PLLA) acid polylactic-co-glycolic-acid (PLGA) scaffold on which neuronal tissue can ex-vivo grow. Further, more the invention provides a method for making cellular vasculature networks and a method for treating a neuronal injury in a subject, by implanting the current composition.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/775,556 filed on Feb. 25, 2013, which claims the benefit of priorityof U.S. Provisional Patent Application No. 61/602,138 filed on Feb. 23,2012. The contents of the above applications are all incorporated byreference as if fully set forth herein in their entirety.

FIELD OF INVENTION

This invention is directed to; inter alia, scaffolds for generating aneuronal tissue and to methods for treating neuronal injuries byimplanting the scaffolds on which neuronal tissue was grown.

BACKGROUND OF THE INVENTION

In the United States alone, approximately 25% of patients in need oforgan transplants die while waiting for a suitable donor. The currentdemands for transplant organs and tissues are far outpacing the supply,and all manner of projections indicate that this gap will continue towiden. Cell transplantation was proposed as an alternative treatment towhole organ transplantation for failing or malfunctioning organs. Forthe creation of an autologous implant, donor tissue is harvested anddissociated into individual cells, and the cells are attached andcultured onto a proper substrate that is ultimately implanted at thedesired site of the functioning tissue. Because many isolated cellpopulations can be expanded in-vitro using cell culture techniques, onlya very small number of donor cells may be necessary to prepare suchimplants. However, it is believed that isolated cells cannot form newtissues, independently. Most primary organ cells are believed to heanchorage-dependent and require specific environments that very ofteninclude the presence of a supporting material to act as a template forgrowth. The success of any cell transplantation therapy therefore relieson the development of suitable substrates for both in-vitro and in-vivotissue culture. Currently, these substrates, mainly in the form oftissue engineering scaffolds, prove less than ideal for applications,not only because they lack mechanical strength, but they also sufferfrom a lack of interconnection channels (Shoufeng Yang, Kah-Fai Leong,Zhaohui Du, and Chee-Kai Chua,. The Design of Scaffolds for Use inTissue Engineering. Part I. Traditional Factors. Tissue Engineering Vol.7 No. 6, 2001).

Tissue engineering applications or even in 3D cell cultures, thebiological cross talk between cells and the scaffold is controlled bythe material properties and scaffold characteristics. In order to inducecell adhesion, proliferation, and activation, materials used for thefabrication of scaffolds must possess requirements such as intrinsicbiocompatibility and proper chemistry to induce molecularbio-recognition from cells. Materials, scaffold mechanical propertiesand degradation kinetics should be adapted to the specific tissueengineering application to guarantee the required mechanical functionsand to accomplish the rate of the new-tissue formation. For scaffolds,pore distribution, exposed surface area, and porosity play a major role,whose amount and distribution influence the penetration and the rate ofpenetration of cells within the scaffold volume, the architecture of theproduced extracellular matrix, and for tissue engineering applications,the final effectiveness of the regenerative process. Depending on thefabrication process, scaffolds with different architecture can beobtained, with random or tailored pore distribution. In the recentyears, rapid prototyping computer-controlled techniques have beenapplied to the fabrication of scaffolds with ordered geometry (CarlettiE, Motta A, and Migliaresi C. Scaffolds for tissue engineering and 3Dcell culture. Methods Mol Biol. 2011; 695:17-39).

Since the publication of Cajal's pioneering studies, it has been clearthat neurons. from the central nervous system (CNS) regenerate poorly,in contrast to those from the peripheral nervous system (PNS).Throughout adult life, olfactory sensory neurons are continuouslyreplenished from progenitor cells of the olfactory neuroepithelium.Furthermore, unlike other peripheral nervous system (PNS) neurons, theseneurons extend axons that reach their final targets in the centralnervous system (CNS)-situated olfactory bulb (OB) (Farbman A I.Olfactory neurogenesis: genetic or environmental controls? TrendsNeurosci. 13:362-5. 1990; Doucette R. Glial cells in the nerve fiberlayer of the main olfactory bulb of embryonic and adult mammals. MicroscRes Tech. 24:113-30. 1993).

Olfactory ensheathing cells (OECs) are a unique glial cell type thatresides in the olfactory bulb and in the olfactory mucosa (Richter M.Westendorf K, Roskams A J. Culturing olfactory ensheathing cells fromthe mouse olfactory epithelium. Methods Mol. Biol. 438:95-102. 2008;Jani H R, Raisman G. Ensheathing cell cultures from the olfactory bulband mucosa. Glia 47:130-7. 2004). OECs envelop olfactory sensory axonsalong their way to the target neurons in the olfactory bulb. Thus, OECshave drawn much attention with respect to CNS axonal regeneration(Moreno-Flores M T, Diaz-Nido J, Wandosell F, Avila J. OlfactoryEnsheathing Glia: Drivers of Axonal Regeneration in the Central NervousSystem? J Biomed Biotechnol. 2:37-43. 2002) and have been proposed tofacilitate this process in the injured CNS (Ramon-Cueto A,Nieto-Sampedro M. Regeneration into the spinal cord of transected dorsalroot axons is promoted by ensheathing glia transplants. Exp Neurol.127:232-44. 1994). Indeed, the regenerative capacity of these cells inthe injured spinal cord and their ability to remyelinate injured spinalcord axons has been confirmed in several studies (Imaizumi T, Lankford KL, Waxman S G, Greer C A, Kocsis J D. Transplanted olfactory ensheathingcells remyelinate and enhance axonal conduction in the demyelinateddorsal columns of the rat spinal cord. J Neurosci. 18:6176-85. 1998).OECs have been suggested to support axon growth in the injured CNS viaexpression of growth factors, such as NGF, NT4/5, NT3, and BDNF (LipsonA C, Widenfalk J, Lindqvist E, Ebendal T, Olson. L. Neurotrophicproperties of olfactory ensheathing glia. Exp Neurol. 180:167-71. 2003;Woodhall E, West A K, Chuah M I. Cultured olfactory ensheathing cellsexpress nerve growth factor, brain-derived neurotrophic factor, gliacell line-derived neurotrophic factor and their receptors. Brain Res MolBrain Res. 88:203-13. 2001).

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a compositioncomprising: A) a porous sponge, B) culture medium, C) nerve growthfactor (NGF), D) olfactory bulb cells, and E) fibronectin, wherein theporous sponge comprises poly-l-lactic acid (PLLA) andpolylactic-co-glycolic-acid (PLGA), wherein NGF is produced by theolfactory bulb cells in the composition, wherein the porous spongecomprises pores having a diameter of 150-800 μm, wherein the porescomprise the olfactory bulb cells, and wherein the olfactory bulb cellsare attached to the surface of the porous sponge and attached within theporous sponge. In another embodiment, the sole source of NGF is OBCsecretion of NGF within the composition of the invention.

In another embodiment, the present invention further provides that theolfactory bulb cells express the NGF receptor p75NTR. In anotherembodiment, the present invention further provides that olfactory bulbcells have neurite extensions. In another embodiment, the presentinvention further provides that the porous sponge comprises at least 85%porosity. In another embodiment, the present invention further providesthat the composition further comprises endothelial cells, fibroblasts,and vasculature networks. In another embodiment, the present inventionfurther provides that the composition is cultured for at least 14 days.

In another embodiment, the present invention further provides a methodfor making cellular vasculature networks, comprising the step ofco-culturing olfactory bulb cells and fibroblasts and/or endothelialcells in a composition such as described herein, wherein the olfactorybulb cells and the fibroblasts and/or endothelial cells are grown on thescaffold of the invention.

In another embodiment, the present invention further provides a methodfor treating a neuronal injury in a subject, comprising the step ofimplanting the composition such as described herein, in a site ofneuronal injury, thereby treating a neuronal injury in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Micrographs showing OECs grown on 3D scaffolds. OB-derived OECswere seeded and cultured on PLLA/PLGA scaffolds prior toimmunofluorescence analysis with Phalloidin TRITC (C and D) to identifyOB cells (gray, white arrows) and a p75NTR (A and B) antibody toidentify OECs (dark gray, light gray arrows). Merge stainings isprovided in E and F. A, C, and E: confocal analysis at low magnification(scale bar=50 μm). B, D, and F: higher magnification. Scale bar=20 μm.

FIG. 2: Micrographs of Viability and proliferation assays of OB-derivedcells grown on 3D scaffolds. OB-derived cells grown on PLLA/PLGAscaffolds were subjected to a live/dead assay using the ethidiumhomodimer (E-H) /calcein AM (A-D) method (gray) and cultured for 48 hr,1 week or 2 weeks prior to analysis. Merge stainings are in I-L. Asnegative control, scaffolds were treated with 0.3% triton X-100 for 1minute prior to the analysis (dark gray). Scale bar=60 μm.

FIG. 3: A graph representing the relative fluorescence of scaffoldsseeded with 200,000 or 500,000 OB-derived cells and were subjected tothe alamar blue proliferation assay at various time points as indicated(n=3).

FIG. 4: Micrographs showing that NGF secretion by OEC induces PC12differentiation on PLLA/PLGA scaffolds. PC12 cells were seeded onPLLA/PLGA scaffolds alone (A-D) or in culture with OEC-containingOB-derived cells (E-H). Co-culture with OB derived cells resulted withrobust (>95%) PC12 differentiation, as indicated by βIII tubulinstaining of long processes emanating from PC12 cells (white arrows). (I,J) Co-culture of purified OB-derived OEC (P75 positive cells) with PC12.OECs are sufficient to induce PC12 differentiation. (K) OB-derivedP75NTR negative (OEC-excluded OB-derived cells) culturing with PC12. Theabsence of OEC in the co-culture resulted in <5% differentiated PC12cells. When the effect of NGF on PC12 cells was blocked by K252a, >95%of PC12 cells did not exhibit neuronal morphology in culture with OBderived cells (M) or in culture with OECs (L), suggesting blocking ofdifferentiation in most of the PC12 cells. (N) While P75NTR stain bothpurified OEC and PC12 cells, βIII tubulin marks only the PC12 cells.Scale bars (μm): A-H, 60; I, 100; J, 20; K, 100; L, 20; M, 100; N, 50.

FIG. 5: Graphs (A: 72 hrs. B: 1 week, C: 2 weeks) showing real-time PCRanalysis of BDNF and NGF gene expression in OB-derived cells cultured on2D and 3D scaffolds (the scaffolds of the invention). RNA fromOB-derived cells grown either as 2D monolayers (black bars) or on 3Dscaffolds (grey bars) was subjected to gene expression analysis at 3time points: 72 hrs, 1 week and 2 weeks post seeding, n=4, student'st-test * p<0.01, ** p<0.05.

FIG. 6: Micrographs (A-D) show in-vitro vascularization analysis onPLLA/PLGA. scaffolds. HUVEC GFP and HFF cells were seeded alone (A, B)or with OB-derived cells (C, D) and cultured for 7 days in HUVEC/HFF/OBmedium. Image presents duplicate scaffolds for each experimental group.White arrows indicate endothelial cells rearrangement. Scale bar=300 μm.

FIG. 7: Are micrographs showing an analysis of distribution of OEC (Cand D) and HUVEC (A and B) in PLLA\PLGA scaffolds. E and F are mergeddistributions. HFF and HUVEC RFP cells were seeded with OB-derived cellson PLLA/PLGA scaffolds. Scale bar=200 μm.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides a compositioncomprising: A) a porous sponge (scaffold), B) culture medium, C) nervegrowth factor (NGF), D) olfactory bulb cells (OBC) attached to theporous sponge, and E) fibronectin, wherein the porous sponge comprisespoly-l-lactic acid (PLLA) and polylactic-co-glycolic-acid (PLGA), andwherein NGF is produced by the olfactory bulb cells attached to thescaffold and/or within the composition. In another embodiment, thepresent invention provides that the porous sponge provides a 3D tissueculture scaffold. In another embodiment, the sole source of NGF in thecomposition of the invention is OBC production and secretion duringculturing with the 3D scaffold as described herein.

In another embodiment, the present invention provides a bioactivescaffold composition such that the bioactive scaffold compositioncontrols the growth of a bioactive regenerative nerve tissue. In anotherembodiment, the present invention provides a bioactive scaffoldcomposition that further supports de-novo, in-vivo neuronal tissuegrowth at a site of implantation.

In another embodiment, the composition comprises a scaffold. In anotherembodiment, the scaffold is a porous sponge. In another embodiment, thesponge is devoid of an organized structure, layer, or network of layers.In another embodiment, the composition is devoid of any layer of alignedfibers. In another embodiment, the scaffold is devoid of any layer ofaligned fibers. In another embodiment, the composition is devoid ofcurved fibers. In another embodiment, the scaffold is devoid of curvedfibers.

In another embodiment, a porous sponge comprises at least 50% porosity.In another embodiment, a porous sponge comprises at least 60% porosity.In another embodiment, a porous sponge comprises at least 70% porosity.in another embodiment, a. porous sponge comprises at least 75% porosity.In another embodiment, a porous sponge comprises at least 80% porosity.In another embodiment, a porous sponge comprises at least 85% porosity.In another embodiment, a porous sponge comprises at least 90% porosity.In another embodiment, a porous sponge comprises at least 92% porosity.In another embodiment, a porous sponge comprises at least 95% porosity.

In another embodiment, a porous sponge comprises pores having a diameterof at least 100 μm. In another embodiment, a porous sponge comprisespores having a diameter of at least 120 μm. In another embodiment, aporous sponge comprises pores having a diameter of at least 150 μm. Inanother embodiment, a porous sponge comprises pores having a diameter of100-900 μm. In another embodiment, a porous sponge comprises poreshaving a diameter of 120-900 μm. In another embodiment, a porous spongecomprises pores having a diameter of 120-850 μm. In another embodiment,a porous sponge comprises pores having a diameter of 150-800 μm. Inanother embodiment, a porous sponge comprises pores having a diameter of200-800 μm. In another embodiment, a porous sponge comprises poreshaving a diameter of 220-750 μm.

In another embodiment, olfactory bulb cells (OBC) is a mixture ofdifferent cell types. In another embodiment, olfactory bulb cells arederived from the olfactory bulb. In another embodiment, olfactory bulbcells comprise fibroblasts, astrocytes and olfactory ensheathing cells(OECs). In another embodiment, olfactory ensheathing cells (OECs) are adistinctive type of glia that secrete neurotrophic factors and formmyelin sheaths around axons projecting from the olfactory mucosa intothe central nervous system olfactory bulb.

In another embodiment, olfactory bulb-derived cells seeded on 3Dscaffolds of the invention exhibit neurotrophic factor expression andpro-angiogenic properties. In another embodiment, the expression of BDNFand NGF genes in cells grown on 3D scaffolds compared to 2D monolayercultures was significantly upregulated by at least 2 fold, 3 fold, 4fold, 5 fold, 6 fold, 8 fold, or by at least 10 fold.

In another embodiment, olfactory bulb cells occupy the scaffold in allthree dimensions. In another embodiment, any cell mentioned hereinoccupies the scaffold in all three dimensions. In another embodiment,olfactory bulb cells occupy the pores. In another embodiment, anolfactory bulb cell resides within a pore. In another embodiment, anolfactory bulb cell resides on the scaffold's surface. In anotherembodiment, olfactory bulb cells axe present both within the pores andon the scaffold's surface.

In another embodiment, cells are syngeneic cells. In another embodiment,cells are allogeneic cells.

In another embodiment, olfactory bulb cells express the NGF receptorp75NTR. In another embodiment, an olfactory bulb cell within a poreexpresses the NGF receptor p75NTR. In another embodiment, olfactory bulbcells express Neurog2. In another embodiment, olfactory bulb cellsexpress NeuN.

In another embodiment, the present invention further provides that thecomposition is cultured for at least 14 days. In another embodiment, thecomposition described herein comprises both poly-l-lactic acid (PLLA)arid polylactic-co-glycolic-acid (PLGA). In another embodiment, thescaffold described herein comprises both poly-l-lactic acid (PLLA) andpolylactic-co-glycolic-acid (PLGA). In another embodiment, PLLA and PLGAare in 1:3 to 3:1 w/w ratio. In another embodiment, PLLA and PLGA are in1:2 to 2:1 w/w ratio. In another embodiment, PLLA and PLGA are in 1:1.5to 1.5:1 w/w ratio. In another embodiment, PLLA and PLGA are in 1:1 w/wratio.

In another embodiment, the composition described herein furthercomprises endothelial cells such as human umbilical vein endothelialcells (HUVEC), fibroblasts such as human foreskin fibroblasts (HFF) orboth. In another embodiment, HUVEC and/or HFF are attached to thescaffold. In another embodiment, the presence of olfactory bulb cellsand HUVEC on a single scaffold results in the formation of vasculaturenetworks. In another embodiment, the presence of olfactory bulb cellsand HFF on a single scaffold results in the formation of vasculaturenetworks. In another embodiment, the presence of olfactory bulb cells,HUVEC and HFF on a single scaffold results in the formation ofvasculature networks. In another embodiment, olfactory bulb cells induceHUVEC and/or HFF to form vasculature network. In another embodiment,cells attached to a scaffold such as described herein comprise neuriteextensions. In another embodiment, OBC attached to a scaffold such asdescribed herein comprises neurite extensions.

In another embodiment, a cell is attached to a scaffold such asdescribed herein for at least 10 days. In another embodiment, a cell isattached to a scaffold such as described herein for at least 14 days. Inanother embodiment, a cell is attached to a scaffold such as describedherein for 10 to 21 days. In another embodiment, a cell is attached to ascaffold such as described herein for 14 to 31 days. In anotherembodiment, an olfactory bulb cell is attached to a scaffold for atleast 14 days.

In another embodiment, a porous sponge-scaffold of the invention isfurther coated with a polymer. In another embodiment, a poroussponge-scaffold of the invention is further coated with an extracellularmatrix protein. In another embodiment, a porous sponge-scaffold of theinvention is further coated with fibronectin. In another embodiment, aporous sponge-scaffold of the invention is further coated. withpolypyrrole. In another embodiment, a porous sponge-scaffold of theinvention is further coated with polycaprolactone. In anotherembodiment, a porous sponge-scaffold of the invention is further coatedwith poly(ethersulfone). In another embodiment, a porous sponge-scaffoldof the invention is further coated withpoly(acrylonitrile-co-methylacrylate) (PAN-MA). In another embodiment, aporous sponge-scaffold of the invention further comprises achemoattractant such as but not limited to laminin-1.

In another embodiment, a composition as described herein furthercomprises fibrin. In another embodiment, a composition as describedherein further comprises thrombin.

In another embodiment, a scaffold such as described herein is 10-160mm³. In another embodiment, a scaffold such as described herein is 10-80mm³. In another embodiment, a scaffold such as described herein is 15-50mm³. In another embodiment, a scaffold such as described herein is asquare. In another embodiment, a scaffold such as described herein is arectangle.

In another embodiment, the three three-dimensional scaffolds describedherein can further include a therapeutic agent. In another embodiment,the therapeutic agent can be any therapeutic agent. In anotherembodiment, the therapeutic agent can be a polypeptide, polypeptidefragment, nucleic acid molecule, small molecule, ribozyme, shRNA, RNAi,antibody, antibody fragment, scFv, enzyme, carbohydrate, or anycombination thereof. In some embodiments, the therapeutic agent can bebrain-derived neurotrophic factor (BDNF), neurotrophic 3 (NT3), nervegrowth factor (NGF), or glial cell-line derived neurotrophic factor(GNDF). The therapeutic agent, in some embodiments, is chondroitinaseABC (chABC) or sialidase. The three-dimensional scaffold can release, inone embodiment, the therapeutic agent for at least 1 day, 1 week, or 1month.

In another embodiment, the scaffold includes a cellular substrate. Inanother embodiment, the cellular substrate is any cellular substrate. Inembodiments, the cellular substrate is a Schwann cell, anoligodendrocyte, an olfactory ensheathing glia (OEG), an oligodendrocyteprogenitor cell (OPC), an embryonic stem cell (ESc), an adult stem cell,an induced pluripotent stem cell, a differentiated ESc anddifferentiated adult Stem cell, an induced pluripotent Stem cell (iPSc),and a macrophage.

In another embodiment, a composition as described herein furthercomprises a material selected from the group consisting of collagen-GAG,collagen, fibrin, PLA, PGA PLA-PGA co-polymer, poly(anhydride),poly(hydroxy acid), poly(ortho ester), poly(propylfumerate),poly(caprolactone), polyamide, polyamino acid, polyacetal, biodegradablepolycyanoacrylate, biodegradable polyurethane and polysaccharide,polypyrrole, polyaniline, polythiophene, polystyrene, polyester,nonbiodegradable polyurethane, polyurea, poly(ethylene vinyl acetate),polypropylene, polymethacrylate, polyethylene, polycarbonate andpoly(ethylene oxide).

In another embodiment, a composition as described herein is cultured forat least 14 days in-vitro, in order to reach baseline proliferationrates.

in another embodiment, a composition as described herein furthercomprises a cell adhesion promoting agent, a proliferation inducer, adifferentiation inducer, an extravasation inducer and/or a migrationinducer. In another embodiment, a composition as described hereinfurther comprises a cell adhesion protein, a growth factor, a cytokine,a hormone, a protease a protease substrate, or any combination thereof.In another embodiment, any substance as described herein is attached tothe scaffold. In another embodiment, any substance as described hereinis embedded within the scaffold. In another embodiment, any substance asdescribed herein is impregnated within the scaffold. In anotherembodiment, a scaffold such as described herein is coated with a gel. Inanother embodiment, a scaffold such as described herein isbiodegradable.

In another embodiment, the porosity of the scaffold is controlled by avariety of techniques known to those skilled in the art. In anotherembodiment, as the porosity is increased, use of polymers having ahigher modulus, addition of suffer polymers as a co-polymer or mixture,or an increase in the cross-link density of the polymer are used toincrease the stability of the scaffold with respect to cellularcontraction.

In another embodiment, the choice of polymer and the ratio of polymersin a co-polymer scaffold of the invention is adjusted to optimize thestiffness/porosity of the scaffold. In another embodiment, the molecularweight and cross-link density of the scaffold is regulated to controlboth the mechanical properties of the scaffold. and the degradation rate(for degradable scaffolds). In another embodiment, the mechanicalproperties are optimized to mimic those of the tissue at the implantsite. In another embodiment, the shape and size of the final scaffoldare adapted for the implant site and tissue type. In another embodiment,scaffold materials comprise natural or synthetic organic polymers thatcan be gelled, or polymerized or solidified (e.g., by aggregation,coagulation, hydrophobic interactions, or cross-linking) into a 3-Dopen-lattice structure that entraps water and/or other molecules, e.g.,to form a hydrogel.

In another embodiment, polymers used in scaffold material compositionsare biocompatible, biodegradable and/or bioerodible and act as adhesivesubstrates for cells. In another embodiment, the structural scaffoldmaterials are non-resorbing or non-biodegradable polymers or materials.The phrase “non-biodegradable polymer”, as used herein, refers to apolymer or polymers which at least substantially (i.e. more than 50%) donot degrade or erode in-vivo. The terms “non-biodegradable” and“non-resorbing” are equivalent and are used interchangeably herein.

In another embodiment, the phrase “biodegradable polymer” as usedherein, refers to a polymer or polymers which degrade in-vivo, andwherein erosion of the polymer or polymers over time occurs concurrentwith or subsequent to release of cells/tissue. The terms “biodegradable”and “bioerodible” are equivalent and are used interchangeably herein.

In another embodiment, scaffold materials comprise naturally occurringsubstances, such as, fibrinogen, fibrin, thrombin, chitosan, collagen,alginate, poly(N-isopropylacrylamide), hyaluronate, albumin, collagen,synthetic polyamino acids, prolamines, polysaccharides such as alginate,heparin, and other naturally occurring biodegradable polymers of sugarunits. In another embodiment, structural scaffold materials are ionichydrogels, for example, ionic polysaccharides, such as alginates orchitosan. Ionic hydrogels may be produced by cross-linking the anionicsalt of alginic acid, a carbohydrate polymer isolated from seaweed, withions, such as calcium cations.

In another embodiment, the scaffolds of the invention are made by any ofa variety of techniques known to those skilled in the art.Salt-leaching, porogens, solid-liquid phase separation (sometimes termedfreeze-drying), and phase inversion fabrication are used, in someembodiments, to produce porous scaffolds.

As used herein, “transplanting” refers to providing the scaffoldsupported cells of the present invention, using any suitable route.Typically, the scaffold supported cells are administered by injectionusing a catheter.

In another embodiment, a culture medium. comprises DMEM/F12, NutrientMix and 7-15% fetal bovine serum. In another embodiment, a culturemedium comprises Neurobasal-A (Invitrogen). In another embodiment, aculture medium comprises 0.5-1.2 mM L-glutamine. In another embodiment,a culture medium comprises 0.1-0.8% methylcellulose. In anotherembodiment, a culture medium comprises 5-15 mM HEPES. In anotherembodiment, a culture medium has a pH of 7.2-7.8. In another embodiment,a culture medium has a pH of 7.4-7.6. In another embodiment, a culturemedium comprises 2-8 μg/ml Gentamycin. In another embodiment, a culturemedium comprises B27-supplement. In another embodiment, a culture mediumcomprises L-15 medium (Invitrogen). In another embodiment, a culturemedium is any medium provided in: Ronald Doucette. Protocols for NeuralCell Culture (2001) which is incorporated herein by reference in itsentirety. In another embodiment, a culture medium is any medium providedin: Doucette, 1984, Doucette, 1986, 11,1; Raisman, 1985; Li et al.,1997; Perez-Bouza et al., 1998; Ramon-Cueto and Nieto-Sampedro, 1994;Ramon-Cueto et al., 1998; Smale et al., 1996; Franklin et al., 1996;Imaizumi et al., 1998; Doucette, 1990; Doucette, 1995; Franklin andBarnett, 1997; Ramon-Cueto and Avila, 1998; Ramon-Cueto and Valverde,1995 which are incorporated herein by reference in their entireties.

In another embodiment, the present invention provided that OB-derivedcells promotes the formation of dense, HUVEC-rich networks of thinvessel-like structures on scaffolds (data not shown), furtherhighlighting the supportive features of OB-derived cells for injuryrepair. In another embodiment, the present invention further provides amethod for making cellular vasculature networks, comprising the step ofco-culturing olfactory bulb cells and endothelial cells in a compositionsuch as described herein, wherein the olfactory bulb cells and theendothelial cells are grown on the scaffold of the invention. In anotherembodiment, OB-derived cells stimulated network formation of endothelialcells grown on the same scaffolds. In another embodiment, 3D scaffoldsof the invention maintained and strengthened the unique therapeuticproperties of embedded OB-derived cells. In another embodiment, thepresent invention provided that OB-derived cells stimulate organizationof endothelial cells into de-novo vasculature networks and thusattracting vascular networks into the spinal cord injury lesion site. Inanother embodiment, the present invention provided that OB-derived cellsgrown on the 3D scaffold of the invention, highly expressed neurotrophicfactors, which then modulated neuronal survival and differentiation. Inanother embodiment, OB-derived cells grown on 3D scaffolds supportedangiogenic behavior, including formation of endothelial cell-basedvessel-like networks. In another embodiment, the 3D PLLA/PLGA scaffoldsdramatically and unexpectedly increased the therapeutic potential ofOB-derived cells for transplantation in patients afflicted withpathologies such as: SCI.

In another embodiment, the present invention further provides a methodfor making cellular vasculature networks, comprising the step ofco-culturing olfactory bulb cells and fibroblasts in a composition suchas described herein, wherein the olfactory bulb cells and thefibroblasts are grown on the scaffold of the invention. In anotherembodiment, the present invention further provides a method for makingcellular vasculature networks, comprising the step of co-culturingolfactory bulb cells, endothelial cells, and fibroblasts in acomposition such as described herein, wherein the olfactory bulb cells,endothelial cells, and the fibroblasts are grown on the scaffold of theinvention. In another embodiment, endothelial cells are HUVEC. Inanother embodiment, fibroblasts are HFF. In another embodiment, a methodfor making cellular vasculature networks is a method of ex-vivovascularizing. In another embodiment, a method for making cellularvasculature networks is a method of ex-vivo vascularizing a neuronal 3Dcell culture within a scaffold. In another embodiment, a method formaking cellular vasculature networks is a method of ex-vivovascularizing a neuronal 3D de-novo tissue culture within a scaffold. Inanother embodiment, a method for making cellular vasculature networkscomprises in-vitro culturing the cells within the scaffold for a periodof at least 1 week. In another embodiment, a method for making cellularvasculature networks comprises in-vitro culturing the cells within thescaffold for a period of at least 10 days. In another embodiment, amethod for making cellular vasculature networks comprises in-vitroculturing the cells within the scaffold for a period of at least 14days.

In another embodiment, the invention further provides a method forneuro-regeneration. In another embodiment, the present invention relatesto the regeneration, reconstruction, repair, augmentation or replacementof a damaged nerve tissue or structure using scaffolds comprising cellsas described herein.

In another embodiment, the invention provides a regenerative cellpopulation containing at least one regenerative cell that when depositedon a scaffold as described herein and implanted into a subject in need(a subject afflicted with neuronal injury), provides a regenerativeeffect for the damaged neuronal tissue that is the subject of thereconstruction, repair, augmentation, or replacement contemplatedherein. In another embodiment, the invention provides that OBC is aregenerative cell population that has the ability to stimulate orinitiate regeneration of a nerve tissue upon implantation into a patientin need. In general, the regeneration of an organ or tissue structure ischaracterized by the restoration of cellular components, tissueorganization and architecture, function, and regulative development. Inaddition, an OBC regenerative cell population grown on a scaffold asdescribed herein minimizes the incompleteness or disorder that tends tooccur at the implantation site. In another embodiment, disorganizationat the site of implantation can manifest itself as increased collagendeposition and/or scar tissue formation, each of which can be minimizedthrough the use of a composition as described herein.

In another embodiment, the invention further provides a method fortreating a neuronal injury in a subject, comprising the step ofimplanting the composition of the invention at a site of neuronalinjury, thereby treating a neuronal injury in a subject. In anotherembodiment, the invention further provides a method for treating aneuronal injury in a subject, comprising the step of implanting ascaffold comprising the cells of the invention at a site of neuronalinjury, thereby treating a neuronal injury in a subject. In anotherembodiment, a scaffold comprising cells such as described herein isimplanted in a lesion cavity. In another embodiment, solubleneurotrophic factors are further administered at the implantation site.In another embodiment, neuronal injury is a peripheral nerve injury. Inanother embodiment, neuronal injury is a CNS injury. In anotherembodiment, neuronal injury is a spinal cord injury.

In another embodiment, a scaffold comprising cells such as describedherein supports neuronal survival and regeneration after spinal cordinjury. In another embodiment, a subject described herein is furthertreated with BDNF, NT-3, and/or VEGF.

In another embodiment, the scaffold to be implanted in a subjectsuffering from a neuronal injury comprises vasculature networks. Inanother embodiment, the scaffold to be implanted in a subject sufferingfrom a neuronal injury comprises OBC and endothelial cells. In anotherembodiment, the scaffold to be implanted in a subject suffering from aneuronal injury comprises OBC and fibroblasts. In another embodiment,the scaffold to be implanted in a subject suffering from a neuronal.injury comprises OBC, endothelial cells, and fibroblasts. In anotherembodiment, the scaffold to be implanted in a subject suffering from aneuronal injury comprises cells having neurite extensions.

The phrase “treating” refers to inhibiting or arresting the developmentof a disease, disorder or condition and/or causing the reduction,remission, or regression of a disease, disorder or condition in anindividual suffering from, or diagnosed with, the disease, disorder orcondition. Those of skill in the art will be aware of variousmethodologies and assays which can be used to assess the development ofa disease, disorder or condition, and similarly, various methodologiesand assays which can be used to assess the reduction, remission orregression of a disease, disorder or condition.

As used herein, the singular forms “a”, “an”, and “the” include pluralforms unless the context clearly dictates otherwise. Thus, for example,reference to “a therapeutic agent” includes reference to more than onetherapeutic agent.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

As used herein, the terms “comprises,” “comprising,” “containing,”“having” and the like can have the meaning ascribed to them in U.S.patent law and can mean “includes,” “including,” and the like;“consisting essentially of or “consists essentially” likewise has themeaning ascribed in U.S. patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic ornovel characteristics of that which is recited is not changed by thepresence of more than that which is recited, but excludes prior artembodiments.

The term “subject” or “patient” refers to an animal which is the objectof treatment, observation, or experiment. By way of example only, asubject includes, but is not limited to, a mammal, including, but notlimited to, a human or a non- human mammal, such as a non-human primate,murine, bovine, equine, canine, ovine, or feline.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disease or condition, e.g., CNS orPNS injury, and/or symptoms associated therewith. Moreover, treatmentincludes the partial or complete regeneration of nerve fibers in asubject. It will be appreciated that, although. not precluded, treatinga disease or condition does not require that the disease, condition, orsymptoms associated therewith be completely eliminated.

As used herein the term “central nervous system disease, disorder, orcondition” refers to any disease, disorder, or trauma that disrupts thenormal function or communication of the brain or spinal cord. The CNSand PNS injuries which can be treated according to the present inventionare diverse and will be easily understood by the skilled person. Withoutlimitation, there may be mentioned brain and spinal cord injuries due toneurosurgery, trauma, ischemia, hypoxia, neurodegenerative disease,metabolic disorder, infectious disease, compression of theintervertebral disc, tumors, and autoimmune disease.

As used herein, the term “therapeutically active molecule” or“therapeutic agent” means a molecule, group of molecules, complex orsubstance administered to an organism for diagnostic, therapeutic,preventative medical, or veterinary purposes. This term includespharmaceuticals, e.g., small molecules, treatments, remedies, biologics,devices, and diagnostics, including preparations useful in clinicalscreening, prevention, prophylaxis, healing, imaging, therapy, surgery,monitoring, and the like. This term can also specifically includenucleic acids and compounds comprising nucleic acids that produce abioactive effect, for example.

In some embodiments, a composition of the invention comprisespharmaceutically active agents. In some embodiments, pharmaceuticallyactive agents are added prior to transplantation. Pharmaceuticallyactive agents include but are not limited to any of the specificexamples disclosed herein. Those of ordinary skill in the art willrecognize also numerous other compounds that fall within this categoryand are useful according to the invention. Examples include a growthfactor, e.g., nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophic 3 (NT3), or glial cell-line derivedneurotrophic factor (GNDF), a steroid, an anti-inflammatory agent, ananalgesic agent, a sedative, a peptidic agent, a biopolymeric agent, anantimicrobial agent, an enzyme (e.g., chondroitinase ABC (chABC) orsialidase), a protein, or a nucleic acid. In a further aspect, thepharmaceutically active agent can be steroids such as betamethasone,dexamethasone, methylprednisolone, prednisolone, prednisone,triamcinolone, budesonide, hydrocortisone, and pharmaceuticallyacceptable hydrocortisone derivatives; non-steroidal antiinflammatoryagents, examples of which include but are not limited to sulfides,mesalamine, budesonide, salazopyrin, diclofenac, pharmaceuticallyacceptable diclofenac salts, nimesulide, naproxene, acetominophen,ibuprofen, ketoprofen and piroxicam, celocoxib, refocoxib, andN-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide; analgesic agentssuch as salicylates; sedatives such as benzodiazapines and barbiturates;antimicrobial agents such as penicillins, cephalosporins, andmacrolides, including tetracycline, chlortetracycline, bacitracin,neomycin, polymyxin, gramicidin, cephalexin, oxytetracycline,chloramphenicol, rifampicin, ciprofloxacin, tobramycin, gentamycin,erythromycin, penicillin, sulfonamides, sulfadiazine, sulfacetamide,sulfamethizole, sulfisoxazole, nitrofurazone, sodium propionate,minocycline, doxycycline, vancomycin, kanamycin, cephalosporins such ascephalothin, cephapirin, cefazolin, cephalexin, cephardine, cefadroxil,cefarnandole, cefoxitin, cefaclor, cefuroxime, cefonicid, ceforanide,cefitaxime, moxalactam, cetizoxime, ceftriaxone, cefoperazone; nucleicacids such as DNA sequences encoding for biological proteins andantisense oligonucleotides; and other pharmacological agents that havebeen shown to promote axonal regeneration such as paclitaxel (TAXOL®).The term also refers to combinations of any of the therapeutic agentsdisclosed herein.

As used herein, the term “biological agent,” “biological molecule,” or“biological therapeutic” is intended to mean a subset of therapeuticagents that are a polypeptide or nucleic acid molecule. In specificembodiments, the biological therapeutic is an agent that induces orenhances nerve growth, e.g., a neurotrophic agent. Examples of usefulneurotrophic agents are ocFGF (acidic fibroblast growth factor), FGF(basic FGF), NGF (nerve growth factor), BDNF (brain derived neurotrophicfactor), CNTF (ciliary neurotrophic factor), MNGF (motor nerve growthfactor), NT-3 (neurotrophin-3), GDNF (glial cell line-derivedneurotrophic factor), NT4/5 (neurotrophin4/5), CM101, HSP-27 (heat shockprotein-27), IGF-I (insulinlike growth factor), IGF-II (insulin-likegrowth factor 2), PDGF (platelet derived growth factor) includingPDGF-BB and PDGF-AB, ARIA (acetylcholine receptor inducing activity),LIF (leukemia inhibitory factor), VIP (vasoactive intestinal peptide),GGF (glial growth factor), and IL-1 (interleukin-1). In a preferredembodiment, the biological therapeutic is NGF or GNDF. In embodiments,the biological therapeutic is an antibody, antibody fragment, or scFVthat induces or enhances nerve growth, e.g., an antibody specific forany of the neurotrophic agents described herein. In other embodiments,the biological therapeutic is a ribozyme, shRNA, or RNAi that induces orenhances nerve growth, e.g., an RNA molecule specific for any of theneurotrophic agents described herein.

As used herein, the term “scaffold” refers to a structure comprising abiocompatible material that provides a surface suitable foradherence/attachment, maturation, differentiation, and proliferation ofcells. A scaffold may further provide mechanical stability and support.A scaffold may be in a particular shape or form so as to influence ordelimit a three-dimensional shape or form assumed by a population ofproliferating cells. All shapes are 3-dimensional and include: films,ribbons, cords, sheets, flat discs, cylinders, spheres, 3-dimensionalamorphous shapes, etc.

As used herein, “biocompatible” means the ability of an object to beaccepted by and to function in a recipient without eliciting asignificant foreign body response (such as, for example, an immune,inflammatory, thrombogenic, or the like response). For example, whenused with reference to one or more of the polymeric materials of theinvention, biocompatible refers to the ability of the polymeric material(or polymeric materials) to be accepted by and to function in itsintended manner in a recipient.

As used herein, “therapeutically effective amount” refers to that amountof a therapeutic agent alone that produces the desired effect (such astreatment of a medical condition such as a disease or the like, oralleviation of a symptom such as pain) in a patient. In some aspects,the phrase refers to an amount of therapeutic agent that, whenincorporated into a composition of the invention, provides apreventative effect sufficient to prevent or protect an individual fromfuture medical risk associated with the transplantation procedure. Aphysician or veterinarian of ordinary skill can readily determine andprescribe the effective amount of the bioactive agent required.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994); Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference. Other general references are provided throughout thisdocument.

Material and Methods Cell Culture

Syngeneic cells from the outer layers of postnatal day-7 (P7) ICR strainmice OBs (nerve fiber and glomerular layers) were isolated, as describedby Ramon-Cueto, but without purification. Cells were cultured in 2Dflasks for 14-17 days, M. DMEM/F12 Nutrient Mix+10% fetal bovine serum(FBS; Biological Industries, Beit-Haemek, Israel). To purify OECs, after14 days in culture the OB-derived cells were sorted by FACS Ariaflowcytometer to P75NTR negative and p75NTR positive (OECs) cells. After14 days in-vitro 10% OEC were measured in the unpurified OB-derived cellpopulation. These results are in line with previous report that 11% ofthe olfactory bulb cells are OECs, 68% are fibroblasts and 21% areastrocytes.

HUVECs (passage 3-7, Clonetics, San Diego, Calif.) were grown on tissueculture plates in EGM-2 medium supplemented with 2% FBS and EGM-2 bulletkit (Cambrex Bio Science, Walkersville). HFF were added tovascularization experiments in order to stabilize the vessels and toimprove the vascularization of the engineered tissues, based on theirpotential to differentiate into smooth muscle cells when co-culturedwith endothelial cells (Caspi O, Lesman A, Basevitch Y, Gepstein A,Arbel G, Habib I H, et al. Tissue engineering of vascularized cardiacmuscle from human embryonic stem cells. Circulation research.100:263-72. 2007.). Primary cultures of HFF cells were prepared fromnewborn foreskin and used until passage 20. HFF cells were cultured inDulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 1%nonessential amino acids (NEAA) (Biological Industries, Beit-Haemek,Israel), and 0.2% β-mercaptoethanol. PC12 cells (a generous gift fromProfessor Alon Chen from the Weizmann Institute of Science) werecultured in DMEM supplemented with 8% horse serum and 8% fetal calfserum (FCS) and incubated at 5% CO₂ at 37° C. To selectively block ofthe effect of nerve growth factor (NGF) on PC12 cells (Koizumi S,Contreras M L, Matsuda Y, Hama T, Lazarovici P, Guroff G. K-252a: aspecific inhibitor of the action of nerve growth factor on PC 12 cells.J Neurosci. 8:715-21. 1988.), the protein kinase inhibitor K252a (200nM, Sigma) was added to co-cultures of 1:10 PC12 and OB-derived cells or1:10 PC12 and OECs cells.

Scaffold Preparation

Porous sponges composed of 50% PLLA (Polysciences, Warrington, Pa., USA)and 50% PLGA (Boehringer-Ingelheim, Ingelheim, Germany) were fabricatedutilizing a particulate leaching technique to achieve pore sizes of212-600 μm and 93% porosity. At total of 0.5 g of PLLA and PLGA (50/50),were dissolved in 10 ml chloroform in a small glass tube to yield asolution of 5% polymer (w/v). A 0.24 ml solution of PLLA/PLGA (50/50)was loaded into Teflon cylinders (diameter=21.5 mm, height=25 mm) packedwith 0.4 g sodium chloride particles with a sieving range of 212-600 μm.The containers were covered for 1 hr, after which the lids were takenoff and the chloroform was evaporated overnight. Scaffolds were removedfrom their mold and placed in a histology cassette. The cassettes wereplaced in a 3-4 L beaker filled with distilled water to leach out thesalt. Scaffolds were then removed from the cassettes, dried and frozenat −80° C. for at least 12 h prior to lyophilization.

Preparation of 3D Tissue Constructs

PLLA/PLGA (50/50) sponges were sliced into squares (5 mm×5 mm) andplaced in 70% ethanol for 1 hr. Scaffolds were then washed inPhospho-buffered saline (PBS) and coated with 20 μg/ml fibronectin(Sigma Aldrich, St. Louis, Mo.) (1 h, room temperature (RT). Prior toseeding, scaffolds were incubated in culture medium for 10 min (RT) andthen partially dried using a vacuum. Pelleted cells were resuspended in5 μl cell culture medium and applied to the scaffolds. After one hour,medium was added and the scaffolds were placed in 37° C. on an XYZorbital shaker. Medium was replaced every other day throughout the 14-and 28-day culturing periods. For vascularization experiments, cellpellets were resuspended in thrombin (50 NIHU/ml, Sigma Aldrich, St.Louis, Mo.) and then mixed with a fibrinogen solution (20 mg/ml, SigmaAldrich, St. Louis, Mo.) at a 1:1 ratio. Thrombin was added to convertthe soluble fibrinogen into insoluble strands of fibrin. Cells were thenadded to the scaffolds and incubated at 37° C. for 30 minutes to allowfor fibrin clot formation. Culturing medium was then added to the platesand the scaffolds were placed on an XYZ orbital shaker. Medium wasreplaced every other day throughout the culturing period.

Immunocytochemistry and Confocal Microscopy

Scaffolds were washed twice with PBS and fixed in a solution of 4% PFAand 4% sucrose (30 min, RT). Scaffolds were then washed twice, andtreated with 0.3% triton X-100 for 10 min, to permeabilize cells.Following triton treatment, scaffolds were washed with PBS and placed ina blocking solution (10% goat serum and 2% BSA in PBS) for 1 hr.Scaffolds were then incubated (4° C., overnight) with primaryantibodies, diluted in blocking buffer. Scaffolds were washed threetimes with PBS, prior to the incubation with the secondary antibodies (2hr, RT). Scaffolds were then washed, immersed in PBS and stored at 4° C.until confocal microscopic analysis. Scaffolds were visualized using theLEICA TCS LSI confocal microscope equipped with PLANAPO 2.0X and5.0X/0.50 LWD lenses.

The NGF receptor p75NTR (1:100, Chemicon, Mississauga, Canada) antibodywas used to identify and sort OEC. The NGF receptors are highlyexpressed in PC12 cells and induce their differentiation intoneuron-like cells. Thus, both PC12 and OECs were positively stained top75NTR, as shown in FIG. 3N. To identify differentiated PC12 from OEC weused the specific neuronal marker βIII tubulin antibody (50 μg/ml,Promega, Madison, Wis.). To measure PC12 differentiation, a cell wasdetermined to he positive for neurite extension if it had at least oneneurite that was longer than the soma diameter of the cell.TRITC-conjugated Phalloidin (1:250, Sigma-Aldrich, St. Louis, Mo.) wasused to stain actin filaments, and DAPI staining (1:1000) was used tovisualize cell nuclei. Antibodies were used according to themanufacturers' recommendations.

Real-Time PCR Analysis

RNA was extracted from cells grown on scaffolds or on tissue cultureplates using the RNeasy Plus Micro Kit (QIAGEN, Germany), according tomanufacturer's protocol. From each experimental and control group, 300ng of RNA was isolated for real-time analysis using a High Capacity cDNAReverse Transcription Kit (Applied Biosystems, Foster City, Calif.,USA). TaqMan assays were performed using gene expression probes,Mm00443039_m1 for NGF, Mm01334042_m1 for BDNF and Mm99999915_g1 forGAPDH as a housekeeping control gene (Applied Biosystems, Foster City,Calif., USA). Results were processed using DataAssist Software (AppliedBiosystems, Foster City, Calif., USA).

In-Vitro Vascularization Analysis

HUVEC and HFF cells were cultured as previously described (Lesman A,Koffler J, Atlas R, Blinder Y J, Kam Z, Levenberg S. Engineeringvessel-like networks within multicellular fibrin-based constructs.Biomaterials. 32:7856-69. 2011). Before seeding, a co-culture of HUVECand HFF cells was prepared at a ratio of 5×10⁵:1×10⁵ cells,respectively. In the indicated experiments, the HUVEC/HFF co-culture wasresuspended with or without 2×10⁵ OB-derived cells in fibrinogen andthrombin, immediately prior to seeding on PLLA/PLGA scaffolds in equalvolumes of HUVEC/HFF/OB culture medium. The suspension was placed on thescaffold and was allowed to be absorbed (1.5 hr. 37° C., 5% CO₂), afterwhich 3 ml of multiculture medium composed of equal volumes ofHUVEC/HFF/OB culture medium was added and replaced every other day.

Viability and Proliferation Assays

To assess cell viability, scaffolds were loaded with calceinacetoxymethyl ester (calcein AM; 1 μmol/L) and ethidium homodimer-1 (4μmol/L) (Sigma-Aldrich, St. Louis, Mo.) for 50 minutes at 37° C., on a3D XYZ shaker. Following dye loading, scaffolds were washed three timeswith PBS and visualized using a confocal microscope. One group ofscaffolds was treated with 0.25% triton x-100 for 1 min prior to theanalysis, and served as a positive dead cell control. Metabolic activitywas assessed using the Alamar blue assay on scaffolds initially seededwith 2×10⁵ or 5×10⁵ OB cells. The medium surrounding the scaffolds wasremoved and replenished with medium containing 10% Alamar blue dye (AbDSerotec, Ireland) on days 1, 3, 6, 8, 10 and 13 post-seeding. Constructswere incubated on an orbital shaker for 6 hr. Samples (100 μl) weretransferred to a clean spectrophotometer assay plate and absorption wasrecorded at 570 and 610 nm. The percentage of reduced dye was calculatedaccording to the manufacturer's recommendations.

Example 1 Seeding OB Primary Cultures on PLLA/PLGA Scaffolds

Olfactory bulbs from P7 mice were dissected and used for the preparationof primary cultures. These cultures were initially grown on 2D platesand were then seeded on PLLA/PLGA porous scaffolds. Cells were grown fora period of 28 days and were then fixed and stained withphalloidin-TRITC and p75NTR specific antibody (FIG. 1). By using lowmagnification microscopy, cell organization around the scaffold's poreswas visualized, with few cells residing in the center of the pores (FIG.1, upper panel). Higher magnification images (lower panel) revealed theorganization and interaction between OECs (positively stained forp75NTR) and phalloidin stained cells in the culture.

The results indicated that following multicellular culturing onscaffolds, the OEC marker p75NTR. was detected around the scaffold pores(FIG. 1). This cell distribution is unique to the present scaffold.Moreover, the scaffold of the present invention unexpectedly maintainedthe viability of neuronal cell in-vitro for prolonged periods andmanaged to support the formation of tissue-like organization within thedescribed artificial settings which are crucial for utilizing thescaffold comprising the cells for implantation procedures. Importantly,the present scaffold unlike previous scaffolds dramatically increasesthe success rate of in-vivo tissue engineering procedures. The presentscaffold maintained the viability of the cells, promoted the formationof thick multicellular constructs in-vitro, promoted tissue organizationand cellular maturation, and promoted differentiation due to itscapacity in allowing for free penetration of both media and nutrients.Furthermore, as mentioned, the present invention's cell-scaffoldarrangements support post-transplantation penetration and viability ofregenerated axons and glia from the scaffold and the host spinal cordinto the scaffold, where they can then integrate with the transplantedOECs.

Example 2 Cells Grown on Scaffolds Remain Viable and Proliferative

In order to analyze the viability of the cells and cell numbers, alive/dead assay and an Alamar Blue assay, were used (FIGS. 2 and 3,respectively). In the live/dead assay, the majority of cells were stillviable at two weeks post-seeding (FIG. 2). As a negative control, thecells on the scaffold were treated with Triton X-100, prior to theanalysis. In this case, the cells appeared dead, as they were positivefor ethidium homodimer (Et-1D) staining. For the quantitativemeasurement of cell numbers, we used Alamar Blue analysis (FIG. 3). Thisanalysis revealed that the cell numbers initially decreases but thenreturns to base level after 14-days in culture. Thus the cell. Thisperiod is necessary in order to reach baseline proliferation rates.

Example 3 OB-Derived Cells Grown on Scaffolds Secrete NGF and InduceNeuronal Differentiation of PC12 Cells

It has been previously shown that OECs express and secrete variousneurotrophic factors, including NGF, BDNF, and GDNF. To examine whetherOEC-containing OB-derived 3D culture affectively secrete NGF, thesecells were co-seeded on PLLA/PLGA scaffolds with pheochromocytoma (PC12)cells. PC12 cell line differentiates to a neuronal lineage in responseto NGF stimulation, manifested by neuronal phenotype and neuronal genesexpression. in this study, differentiation of PC12 cells served as anindex for NGF secretion by olfactory bulb cells. To identify PC12 andOECs, the co-culture was stained with P75NTR (expressed by both PC12 andOEC) and βIII-tubulin (mark only PC12), as shown in FIG. 3N. In theabsence of OB-derived cells, PC12 cells appeared round, withoutprocesses and expressed both p75NTR and βIII-tubulin (FIG. 4A-D).However, when seeded together with OB-derived cells (FIG. 4E-H), PC12cells took on a neuronal-like morphology with long processes emanatingfrom their cell bodies (FIG. 4H, white arrowheads). PC12 cells acquiredsimilar morphology when cultured only with purified (p75NTR positivecells) OECs (FIG. 4I, J). PC12 differentiation in the presence ofOB-derived or OECs was robust and occurred in more than 95% of thecells. Less than 5% of the PC12 cells exhibited the differentiatedphenotype in culture with OEC-excluded OB cells (p75NTR negative cells,FIG. 4K). Previous studies have shown that NGF is involved in theinduction of PC12 differentiation (Doucette R. Glial cells in the nervefiber layer of the main olfactory bulb of embryonic and adult mammals.Microsc Res Tech. 24:113-30. 1993). Under the current conditions it wasfound that the culture medium of OB-derived cells contains secreted NGF(27.6 pg/ml±7.89 SE). To address this, the role of NGF was examined byusing K252a which selectively block of the effects of NGF on PC12 cells(Koizumi S, Contreras M L, Matsuda Y, Hama T, Lazarovici P, Guroff G.K-252a: a specific inhibitor of the action of nerve growth factor on PC12 cells. J Neurosci. 8:715-21, 1988). When K252a was added to theculture medium of PC12 and OB-derived cells (FIG. 3M) or purified OEC(FIG. 4L). more than 95% of PC12 cells did not acquire neuronal-likemorphology. Taken together, these results suggest that OECs effectivelysecrete bioactive growth factors such as NGF to the medium which caneffect neuronal differentiation.

Thus the secretion of NGF by OEC was examined, when cultured on 3Denvironment. After 14 days in culture, in comparison to the 2D cultures,the expression levels of NGF genes were significantly higher. Inaddition, 3D-cultured OB derived cells and OECs in particular inducedrobust differentiation of PC12. This effect was blocked when the PC12response to NGF was selectively inhibited, further demonstrating thecritical role of NGF in PC12 differentiation. Taken together, theseresults demonstrate in-vitro NGF secretion by OECs on PLLA/PLGA 3Dscaffolds and offer an advantageous tool for transplantation in spinalcord injury site.

Example 4 Expression Levels of NGF and BDNF are Dependent on the CultureSpatial and Temporal Organization

Quantitative RT-PCR analysis was performed on RNA samples isolated fromcells grown on PLLA/PLGA scaffolds and compared to cells grown in amonolayer. At 72 hr post-seeding, cells grown in monolayers exhibitedhigher levels of both NGF and BDNF when compared to those grown on 3Dscaffolds. After one week in culture, similar expression levels of thetwo genes were measured in both culturing setups. However, when comparedto cells grown in 2D monolayers after 14 days, the expression level ofNGF and BDNF genes in cells grown on 3D scaffolds was 350% and 30%higher, respectively (FIG. 5).

Example 5 OB-Derived Cells Modulate HUVEC-Re-Organization on 3DScaffolds

In order to assess the ability of OB-derived cells to promote 3Dvascular network. formation in-vitro, OB-derived cells were seededtogether with HUVEC-GFP and HFF cells on PLLA/PLGA scaffolds andcultured in a medium mix composed of equal volumes of the specificculture medium for each cells (described in “materials and methods”).HUVEC cells cultured in the absence of OB-derived cells remainedunorganized (FIG. 6A, B). However, after seven days in culture, in thepresence of OB-derived cells, HUVEC-GFP cells organized in an orderlyvascular network (FIG. 6 C, D). The interaction between OECs and HUVECcells was also analyzed by staining OECs seeded with HUVEC-RFP and HFFcells on PLLA/PLGA scaffolds, with anti-p75NTR antibodies (FIG. 7). OECswere primarily localized at the periphery of the scaffold, whereas HUVECcells were homogenously dispersed throughout the entire scaffold.

The present findings showed that OB-derived cells promoted the formationof dense, HUVEC-rich networks of thin vessel-like structures on bothPLLA/PLGA and SIS scaffolds (data not shown), further highlighting thesupportive features of OB-derived cells for injury repair. These resultsclearly indicate, for the first time, that OB-derived cells stimulateorganization of endothelial cells into de-novo vasculature networks andmay explain their ability to attract vascular networks into the spinalcord injury lesion site.

Taken together, the present study demonstrates the feasibility ofseeding and culturing OB-derived cells on 3D scaffolds. The 3D scaffoldsetting dramatically enhanced neurotrophic factors expression, which aresuggested to then modulate neuronal survival and differentiation. Inaddition, OB-derived cells grown on 3D scaffolds supported viability,angiogenic behavior, including formation of endothelial cell-basedvessel-like networks. OB-derived cells cultures on 3D PLLA/PLGAscaffolds are ready to he utilized therapeutically and the presentfindings will enable the design of multi-cellular OB-derivedcell-enriched tissues suitable for transplantation in SCI patients.

What is claimed is:
 1. A method for treating a neuronal injury in asubject, comprising the step of implanting an ex-vivo vascularizedimplant in a site of neuronal injury, said ex-vivo vascularized implantcomprises: a porous sponge comprising poly-l-lactic acid (PLLA) andpolylactic-co-glycolic-acid (PLGA); olfactory bulb cells attached to thesurface of said porous sponge and within said porous sponge; anendogenous nerve growth factor (NGF) de-novo produced by said olfactorybulb cells; and vasculature formed in-vitro within said porous sponge,thereby treating a neuronal injury in a subject.
 2. The method of claim1, wherein said olfactory bulb cells express the NGF receptor p75NTR. 3.The method of claim 1, wherein said olfactory bulb cells comprisefibroblasts, astrocytes and olfactory ensheathing cells.
 4. The methodof claim 1, wherein said ex-vivo vascularized implant further comprisesan endothelial cell, a fibroblast, or both.
 5. The method of claim 1,wherein said ex-vivo vascularized implant further comprises fibronectin,fibrin, thrombin, or any combination thereof.
 6. The method of claim 1,wherein said ex-vivo vascularized implant further comprisesbrain-derived neurotrophic factor (BDNF) de-novo produced by saidolfactory bulb cells.
 7. A method for making a neuronal ex-vivovascularized implant, comprising the step of co-culturing cells on a 3Dscaffold in the presence of culture medium, said cells comprisesolfactory bulb cells and endothelial cells, said 3D scaffold comprisespoly-l-lactic acid (PLLA) and polylactic-co-glycolic-acid (PLGA), saidneuronal ex-vivo vascularized implant produces nerve growth factor(NGF), thereby making a neuronal ex-vivo vascularized implant.
 8. Themethod of claim 7, wherein said ex-vivo vascularized implant expressesthe NGF receptor p75NTR.
 9. The method of claim 7, wherein saidolfactory bulb cells comprise fibroblasts, astrocytes and olfactoryensheathing cells.
 10. The method of claim 7, wherein said 3D scaffoldfurther comprises fibronectin, fibrin, thrombin, or any combinationthereof.
 11. The method of claim
 7. wherein said ex-vivo vascularizedimplant further comprises brain-derived neurotrophic factor (BDNF)de-novo produced by said olfactory bulb cells.